Coronary artery disease is a major cause of mortality and disability in the United States and worldwide, and accounts for rising health costs. Clinically it is evident that plaque rupture, the most important cause of coronary thrombosis, is often associated with myocardial infarction and death. Many of the endogenous ligands that play a key role in plaque rupture are still not well understood. A variant of the extracellular matrix protein fibronectin containing the alternatively-spliced extra domain A (EDA+-FN) is absent in healthy arteries, but expressed in the atherosclerotic arteries of humans, suggesting a role in the pathophysiology of atherosclerosis. Our recent study using a specific inhibitor suggests that EDA+-FN aggravates ischemia/reperfusion brain injury via toll-like-receptor 4 (TLR4) pathway. Together these findings provide a compelling rationale to test the novel role of EDA+-FN and TLR4 signaling in modulating early and advanced atherosclerosis. In Aim 1, we will test the hypothesis that EDA+-FN exacerbates early atherosclerosis via TLR4 signaling in apolipoprotein E-deficient (ApoE-/-) mice. Further, we will define the role of endothelial cell TLR4 in exacerbating early atherosclerosis. In Aim 2, we will test the hypothesis that TLR4 signaling contributes to plaque vulnerability during advanced atherosclerosis in ApoE-/- mice. Further, we will test the hypothesis that EDA+-FN promotes plaque vulnerability via TLR4 signaling. In Aim 3, we will define the role of endothelial cell EDA+-FN in atherosclerosis. As a preclinical approach, we will test the hypothesis that blocking EDA+-FN with monoclonal antibodies will inhibit atherosclerotic lesion progression in ApoE-/- mice. The proposed studies will use multidisciplinary innovative approaches, including several novel genetic mouse strains, and a novel intravital method to define the mechanisms by which EDA+-FN modulates atherosclerosis. The proposal is significant and may have important clinical implications because the studies designed herein will identify a novel endogenous ligand and unravel new pathway that modulate key atherogenic events in early and advance lesion development.